1. Contributions of KRAS and RAL in Non–Small-Cell Lung Cancer Growth and Progression 
Sunny Guin, PhD, Yuanbin Ru, PhD, Murry W. Wynes, PhD, Rangnath Mishra, PhD, Xian Lu, MS, Charles Owens, MS, Anna E. Barόn, PhD, Vihas T. Vasu, PhD, Fred R. Hirsch, MD, PhD, Jeffrey A. Kern, MD, Dan Theodorescu, MD, PhD 
Journal of Thoracic Oncology 
Volume 8, Issue 12, Pages (December 2013)
DOI: /JTO
Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

2. FIGURE 1
RALA and B cytoplasmic and cell membrane immunohistochemical staining in the NSCLC patient tumor samples. H-score was used to measure the staining for RAL protein expression (see Supplementary Materials and Methods section, Supplemental Digital Content 1, The average H-score of the triplicate cores per patient was highly correlated to the core with the highest score from the same patient, thus further analyses were performed using the maximum H-score. A and C, Show typical examples of (i) low or no cytoplasmic and membranous protein; (ii) high membranous and low cytoplasmic; (iii) high cytoplasmic and high membranous staining for RALA and RALB. B and D, Show three graphical depictions of RALA and B membrane and cytoplasmic staining in NSCLC specimens. NSCLC, non–small-cell lung cancer.
Journal of Thoracic Oncology 2013 8, DOI: ( /JTO )
Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

3. FIGURE 2
RALA and B protein and mRNA expression stratify NSCLC patient survival. Overall NSCLC patient survival as a function of (A) RALA and (B) RALB membrane expression as evaluated by IHC. C, Patient survival in NSCLC data sets GSE8894 and GSE11969 (Supplementary Table 3) as a function of RALA (i) mRNA expression or (ii) risk score (see Materials and Methods section). D, Overall patient survival in NSCLC data set GSE4716_GPL3694 as a function of RALB expression. IHC, immunohistochemistry; HR, hazard ratio; NSCLC, non–small-cell lung cancer.
Journal of Thoracic Oncology 2013 8, DOI: ( /JTO )
Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

4. FIGURE 3
RALA and RALB expression and activity in human NSCLC cell lines. A, RALA and RALB expression in 14 NSCLC cell lines by Western blot. Cell lines with KRAS mutation indicated with asterisks. B, Densitometric quantification of RALA and RALB expression shows their expression is higher in KRAS mutant NSCLC cell lines (p < 0.05, Student’s t test). C, RALA and RALB activation levels in the 14 NSCLC cell lines. Cell lines with KRAS mutation indicated with asterisks. D, Densitometric analysis revealed higher RALA activation in KRAS mutant NSCLC cell lines (p < 0.05, Student’s t test). No relationship to KRAS was found with RALB. E, Knockdown of RAL GTPase in H2122 cells after transient transfection with siRNA against RALA (siRALA) and B (siRALB). Cells were lysed 3, 6, 9, 12, 15, and 18 days after transfection, and RAL GTPase knockdown was determined by Western blot. F, Loss of RALA, B or A+B reduced subcutaneous tumor growth in mice. H2122 cells were transfected with siRNA against RALA (siRALA), B (siRALB), and A + B (siRALA+B), injected in mice and studied for in vivo tumor growth (see Materials and Methods section). Luciferase siRNA transfected cells were used as control (siCTL). *p < 0.05 by Student’s t test. NSCLC, non–small-cell lung cancer; WT, wild-type; siRNA, small interfering RNA.
Journal of Thoracic Oncology 2013 8, DOI: ( /JTO )
Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

5. FIGURE 4
The role of RAL and KRAS in tumor growth. A, KRAS expression in H2228 cells stably transfected with empty vector (CTL), KRAS WT, KRAS G12C and G12V mutants (KRAS G12C and KRAS G12V) detected by Western blot. B, KRAS activation in the engineered H2228 cell lines as determined by RAS activation enzyme-linked immunosorbent assay (see Materials and Methods section). *p < 0.05 by Student’s t test. C, RALA and B activation observed by Western blot in H2228 cells transfected with empty vector (CTL), KRAS WT, and KRAS G12C and G12V mutants (KRAS G12C and KRAS G12V) (see Materials and Methods section). D, Densitometric analysis of RALA and B activation observed in (C). Asterisk and pound indicate p < 0.05 for RALA and B by Student’s t test. E, RALA and B loss observed by Western blot after transfection of engineered H2228 cell lines with siRNA targeting both RALs (siRALA+B). A luciferase siRNA transfected cells are used as control (siCTL). F, Anchorage independent growth of engineered H2228 cell lines after loss of RAL GTPase (*p < 0.05 by Student’s t test). WT, wild-type; siRNA, small interfering RNA.
Journal of Thoracic Oncology 2013 8, DOI: ( /JTO )
Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

6. FIGURE 5
RAL and KRAS mRNA expression and risk score stratify overall and recurrence-free survival in NSCLC patients. A, Recurrence-free survival in NSCLC data set GSE8894 as a function of RALA mRNA expression in patients with high KRAS mRNA expression. B, Patient overall survival in NSCLC data set GSE11969 as a function of KRAS risk score in patients with high RALA risk score. C, Patient overall survival in NSCLC data set GSE4716_GPL3694 as a function of RALB mRNA expression in patients with high KRAS mRNA expression. D, Patient overall survival in NSCLC data set GSE4716_GPL3694 as a function of KRAS mRNA expression in patients with high RALB mRNA expression. NSCLC, non–small-cell lung cancer; HR, hazard ratio.
Journal of Thoracic Oncology 2013 8, DOI: ( /JTO )
Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions